美国麻省大学医学院Wen Xue研究组利用prime editing对长基因组序列进行删除和替换。该研究于2021年10月14日在线发表于国际一流学术期刊《自然—生物技术》。

研究人员优化了用于创建精确基因组缺失的prime editing（PE）工具，并在没有外源DNA模板的情况下，直接用所需的序列（最多60bp）替换从~1kb到~10kb的基因组片段。通过将Cas9核酸酶与逆转录酶（PE-Cas9）连接，并与两个针对互补DNA链的PE引导RNA（pegRNA）相结合，研究人员通过使用这种基于PE-Cas9的删除和修复（PEDAR）方法，实现了对目标序列的精确和特定的删除和修复。PEDAR在报告系统和内源性位点的表现优于其他基因组编辑方法，有效地创造了大量精确的基因组改变。在小鼠的酪氨酸血症模型中，PEDAR删除了Fah基因内1.38kb的致病性插入，并精确地修复了缺失结点来恢复肝脏中FAH的表达。

据悉，占人类致病性突变约14%的基因组插入、复制和插入/删除（indel）不能被目前的基因编辑方法准确或有效地纠正，特别是那些涉及较大改变（>100bp）的改变。

附：英文原文

Title: Deletion and replacement of long genomic sequences using prime editing

Author: Jiang, Tingting, Zhang, Xiao-Ou, Weng, Zhiping, Xue, Wen

Issue&Volume: 2021-10-14

Abstract: Genomic insertions, duplications and insertion/deletions (indels), which account for ~14% of human pathogenic mutations, cannot be accurately or efficiently corrected by current gene-editing methods, especially those that involve larger alterations (>100base pairs (bp)). Here, we optimize prime editing (PE) tools for creating precise genomic deletions and direct the replacement of a genomic fragment ranging from ~1kilobases (kb) to ~10kb with a desired sequence (up to 60bp) in the absence of an exogenous DNA template. By conjugating Cas9 nuclease to reverse transcriptase (PE-Cas9) and combining it with two PE guide RNAs (pegRNAs) targeting complementary DNA strands, we achieve precise and specific deletion and repair of target sequences via using this PE-Cas9-based deletion and repair (PEDAR) method. PEDAR outperformed other genome-editing methods in a reporter system and at endogenous loci, efficiently creating large and precise genomic alterations. In a mouse model of tyrosinemia, PEDAR removed a 1.38-kb pathogenic insertion within the Fah gene and precisely repaired the deletion junction to restore FAH expression in liver.

DOI: 10.1038/s41587-021-01026-y

Source: https://www.nature.com/articles/s41587-021-01026-y